Method to induce neovascular formation and tissue regeneration

ABSTRACT

The present invention relates, e.g., to a method for inducing arteriogenesis, lymphangiogenesis, vasculogenesis, or cardiomyogenesis, and/or for inducing mitosis or proliferation of a smooth muscle cell, a skeletal muscle cell, or a cardiomyocyte, comprising administering to a cell or tissue in need thereof a dose of a polynucleotide that encodes a vascular endothelial growth factor (VEGF), or that encodes a polypeptide comprising an active site of the VEGF. The coding sequence is operably linked to an expression control sequence; and the dose is sufficient to induce arteriogenesis, lymphangiogenesis, vasculogenesis, or cardiomyogenesis, and/or to induce mitosis or proliferation of a smooth muscle cell, a skeletal muscle cell, or a cardiomyocyte. In preferred embodiments of the invention, the method is a method of tissue regeneration, particularly of cardiomyogenesis; and the polynucleotide (or a polypeptide encoded by such a polynucleotde) is administered into the myocardium.

This application is a continuation-in-part of PCT/US02/14508, filed May13, 2002, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates, e.g., to a method for inducing arteriogenesis,lymphangiogenesis, vasculogenesis, or cardiomyogenesis, or for inducingmitosis or proliferation of a smooth muscle cell, a skeletal musclecell, or a cardiomyocyte, using a Vascular Endothelial Growth Factor(VEGF). For example, a gene therapy method is described for in vivolocalized induction of neovascular formation or tissue regeneration inmammals utilizing VEGF.

BACKGROUND OF THE INVENTION

Ischemic heart disease is the main cause of morbidity and mortality. Theepidemiological and socio-economical impact of coronary heart disease isremarkable. This disease causes million of deaths all over the world.See Murray, et al., Lancet, 349:1269-1276 (1997). In developedcountries, it has been estimated that 5.3 million deaths attributable tocardiovascular disease occurred in 1990, whereas the correspondingfigure for the developing countries ranged between 8 to 9 million(showing a relative excess of 70%). See Reddy, et al., Circulation,97:596-601 (1998). In Argentina, ischemic heart disease is the firstcause of mortality showing an incidence of around 30%, trend which tendsto remain stable since 1980. For the population over 65 years, this ratereaches almost 40%. See Programa Nacional de Estadísticas de Salud,Series 5, Number 38, Ministerio de Salud y Acción Social, RepúblicaArgentina (December 1995).

Despite recent advances in prevention and treatment of ischemic heartdisease, there are many patients who are still symptomatic and cannotbenefit from conventional therapy. Administration of growth factors thatpromote neovascular formation and growth, such as fibroblast growthfactors (FGFs) and VEGF, appear as a novel and promising alternative forthese patients. This mode of treatment is called therapeuticangiogenesis. See Henry, B. M. J., 318:1536-1539 (1999).

VEGF is a protein expressed by skeletal muscle cells, smooth musclecells, ovarian corpus luteum cells, tumor cells, fibroblasts andcardiomyocytes. Unlike other mitogens, VEGF is a secreted growth factor.See Thomas, J. Biol. Chem, 271:603-606 (1996); Leung, et al., Science,246:1306-1309 (1989). The human VEGF gene is expressed as differentisoforms, secondary to post-transcriptional alternative splicing. Innon-malignant human tissues, four VEGF isoforms are expressed, withdifferent numbers of amino acids (121, 165, 189, 206) and with amolecular weight ranging from 34 to 46 kD. See Tischer, et al., J. Biol.Chem., 266:11947-11954 (1991); Ferrara, et al., J. Cell. Biochem.,47:211-218 (1991).

VEGF specific receptors are VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1) andVEGFR-3 (flt-4). See De Vries, et al., Science, 255:989-991 (1992);Terman, et al., Biochem. Biophys. Res. Commun., 187:1579-1586 (1992);Gallant, et al., Genomics, 13:475-478 (1992). Due to the apparentrestricted and confined localization of VEGF receptors to vascularendothelial cells, this growth factor has been described as the mostspecific mitogen for these cells. It has been proposed that VEGF is notbioactive on non-endothelial cells. See Jakeman, et al., J. Clin.Invest., 89:244-253 (1992); Ferrara, et al., Endocr. Rev., 18:4-25(1997); Thomas, et al., supra (1996). However, recent studies havereported mitogenic effects of VEGF on some non-endothelial cell types,such as retinal pigment epithelial cells, pancreatic duct cells andSchwann cells. See Guerring et al., J. Cell. Physiol., 164:385-394(1995); Oberg-Welsh et al., Mol. Cell. Endocrinol., 126:125-132 (1997);Sondell et al., J. Neurosci., 19:5731-5740 (1999). Moreover, VEGFreceptors have been found in other cells, such as hematopoietic stemcells, endocardial cells and even cultured rat cardiomyocytes, whereVEGF has been shown to activate the mitogen-activated protein kinasecascade. See Asahara et al., Science, 275:964-967 (1997); Partanen etal., Circulation, 100:583-586 (1999); Takahashi et al., Circ. Res.,84:1194-1202 (1999).

Therapeutic administration of VEGF is a significant challenge. VEGF canbe administered as a recombinant protein (protein therapy) or byVEGF-encoding gene transfer (gene therapy). See Safi, et al., J. Mol.Cell. Cardiol., 29:2311-2325 (1997); Simons, et al., Circulation,102:E73-E86 (2000).

Protein therapy has several disadvantages. The extremely short mean-lifeof angiogenic proteins (e.g. VEGF) conditions therapy to theadministration of high or repeated doses to achieve a noticeable effect.See Simons, et al., supra (2000); Takeshita, et al., Circulation,90:11228-234 (1994). Furthermore, intravenous administration of highdoses of VEGF protein is known to induce severe or refractoryhypotension. See Henry, et al., J. Am. Coll. Cardiol., 31:65A (1998);Horowitz, et al., Arterioscl. Thromb. Vasc. Biol., 17:2793-2800 (1997);López, et al., Am. J. Phisiol., 273:H1317-1323 (1997). To avoid thesedisadvantages, gene therapy (e.g. DNA encoding for VEGF) has beenproposed. See Mack, et al., J. Thorac. Cardiovasc. Surg., 115:168-177(1998); Tio, et al., Hum. Gene Ther., 10:2953-2960 (1999).

Gene therapy can be compared to a drug slow-delivery system. The geneencoding for the agent of interest is transported into cells in vehiclescalled vectors (e.g. plasmids, viruses, liposomes). Cell mechanismsspecialized in protein synthesis perform the production and localizedrelease of the final product. See Crystal, Science, 270:404-410 (1995).In addition, it should be noted that in the case of plasmids the geneproduct is synthesized for a discrete period of time. This time isusually about two weeks. According to experimental studies, sustainedexpression during this limited period of time is necessary andsufficient to trigger the angiogenic process. Based on these advantages,several research groups have studied the therapeutic effects of genetherapy using angiogenic factors in experimental models of heart andlimb ischemia. These approaches have yielded promising results. SeeMagovern, Ann. Thorac. Surg., 62:425-434 (1996); Mack, et al., supra(1998); Tio, et al., supra (1999); Walder, et al., J. Cardiovasc.Pharmacol., 27:91-98 (1996); Takeshita, et al., Lab. Invest., 75:487-501(1996); Mack, et al., J. Vasc. Surg., 27:699-709 (1998); Tsurumi, etal., Circulation, 94:3281-3290 (1996). Gene therapy has achieved theexpected effects without the shortcomings associated with proteintherapy. However, adenoviral gene therapy may induce inflammatory orimmune reactions, especially after repeated doses. This type of therapyhas been related also to high risk systemic immune response syndrome.These circumstances limit significantly the clinical use of thistherapy. See Gilgenkrantz, et al., Hum. Gene Ther., 6:1265-1274 (1995);Dewey, et al., Nat. Med., 5:1256-1263 (1999); Wersto, et al., J. Virol.,72:9491-9502 (1998); Hollon, Nat. Med., 6:6 (2000), Chan, et al., Nat.Med., 5:1143-1149 (1999); Byrnes, et al., J. Nerosci., 16:3045-3055(1996). According to recent studies, plasmid gene therapy does not havethese disadvantages and can be administrated safely in repeated doses.See Simons, et al., supra (2000).

Systemic administration of VEGF has been associated with undesiredangiogenesis in peripheral tissues. See Folkman, Nat. Med., 1:27-31(1995); Liotta, et al., Cell, 64:327-336 (1991); Lazarous, et al.,Circulation, 94:1074-1082 (1996); Ferrara, Breast Cancer Res. Treat.,36:127-137 (1995); Ferrara, Lab. Invest., 72:615-618 (1995); Aiello, etal., N. Eng. J. Med., 331:1480-1485 (1994); Adams, et al., Am. J.Ophthalmol., 118:445-450 (1994); Inoue, et al., Circulation,98:2108-2116 (1998); Simons, et al., supra (2000). The risk of systemicexposure is probably more related to the route of administration than tothe nature of therapy (gene or protein) utilized. In comparison withintravascular delivery, local (e.g. intramyocardial) administrationreduces the risk of systemic exposure and undesired peripheralangiogenesis. See Simons, et al., supra (2000).

At the present, it has been demonstrated that VEGF induces angiogenesisin vivo. It has not been reported yet that VEGF induces the formation ofblood vessels with a smooth muscle layer. See Mack, et al., supra(1998); Tio, et al., supra (1999). Moreover, it has been postulated thatVEGF prevents the neoformation of vascular smooth muscle. See Asahara,et al., Circulation, 91:2793-2801 (1995). Smooth muscle plays asignificant role in the regulation of vascular function. Its presence atthe media layer of blood vessels represents an adaptative advantagesince it is involved in the vasomotor tone regulation. Vascular smoothmuscle maintains a basal vascular tone and permits self-regulation uponvariations on blood flow and pressure. It has been suggested that theabsence of smooth muscle layer is related to vessel collapse. See“Angiogenesis and Cardiovascular Disease”, Ware, Ed. (Oxford UniversityPress Inc., New York, USA., 1999), p. 258-261.

Acute myocardial infarction is the consequence of coronary heart diseasewith the worst short and long-term prognosis. See Bolognese, et al., Am.Heart J., 138:579-83 (1999); Mehta, et al., Herz, 25:47-60 (2000);Hessen, et al., Cardiovasc. Clin., 20:283-318 (1989); Jacoby, et al., J.Am. Coll. Cardiol., 20:736-744 (1992); Rosenthal, et al., Am. Heart J.,109:865-876 (1985). This condition results frequently in a significantloss of myocardial cells, reducing the contractile muscle mass. It isknown in the art that cardiomyocytes of human and human-like speciespreserve their ability to replicate DNA. See Pfizer, et al., Curr. Top.Pathol., 54:125-168 (1971). Recently, it has been informed that somehuman cardiomyocytes can enter into M (mitotic) phase. However, thisphenomenon occurs in a very small proportion of total cardiomyocytepopulation and under certain pathological conditions. So far, thisphenomenon has only been noted in myocardial infarction and end-stagecardiac failure. See Beltrami et al., N. Eng. J. Med., 344: 1750-1757(2001); Kajstura, et al., Proc. Natl. Acad. Sci. USA, 95:8801-8805(1998). There is no conclusive evidence in all these instances thatcardiomyocytes divide into daughter cells.

The inability of cardiomyocytes to replicate properly precludes thereplacement of myocardial tissue after injury in upper animal species.Under this scenario, myocardial function is diminished because theinfarcted area is replaced by fibrotic tissue without contractilecapacity. In addition, the remaining cardiomyocytes become hypertrophicand develop polyploid nuclei. See Herget, et al., Cardiovasc. Res.36:45-51 (1997); “Textbook of Medical Physiology”, 9th Ed., Guyton etal., Eds. (W. B. Saunders Co, USA, 1997).

Attempts have been made to restore myocardial cell loss with othercells, such as autologous satellite cells and allogenic myoblasts. Theresults of these attempts are not conclusive. See Dorfman, et al., J.Thorac. Cardiovasc. Surg., 116:744-751 (1998); Murry, et al., J. Clin.Invest., 98: 2512-2523 (1996); Leor, et al., Circulation, 94 Suppl.II:II-332-II-336 (1996); Li et al., Circ. Res., 78:283-288 (1996). Morerecently, it has been suggested that pluripotent stem cells and bonemarrow derived angioblasts might restore infarcted myocardial tissue andinduce even neovascular formation. However, the efficiency of thesemethods in upper mammals has not been demonstrated yet. See Orlic, etal., Nature, 410:701-705 (2001); Kocher, et al., Nat. Med., 7:430-436(2001). An ideal method should induce cardiomyocyte division originatingdaughter cells and neovascular formation in myocardial tissue. Thisprocedure would restore tissue loss with autologous myocardial tissueand increase simultaneously myocardial perfusion. A method like thiswould reduce the morbility and mortality rates associated to leftventricular remodeling, myocardial infarction and ischemic heartdisease. See Bolognese, et al., supra (1999).

Likewise, the failure of cardiomyocytes to replicate properly difficultsadaptative hyperplasia (i.e. cell number increasing) as a response toother pathological conditions. In these cases, the adaptative responseof human and porcine cardiomyocytes is to increase cell volume andnuclear DNA content. Therefore, in certain pathologies (e.g.hypertensive heart disease, dilated cardiomyopathy) cardiomyocytes arealso markedly hypertrophic and polyploid. See Pfizer, Curr. Top.Pathol., 54:125-168 (1971); Adler, et al., J. Mol. Cell. Cardiol.,18:39-53 (1986). In most cases, cell adaptation is insufficient.Besides, the cellular demand for oxygen and nutrients increases asmyocardial hypertrophy progresses. In consequence, the increased demandsimpair subendocardial perfusion even in the absence of coronaryocclusion. Finally, the combination of these factors leads to myocardialfunction detriment. See “Textbook of Medical Physiology”, 9^(th) Ed,supra. An ideal method should induce mitosis on hypertrophic andpolyploid cells. This method should result in smaller andbetter-perfused daugther cells thus reducing the progression ofcardiomyopathy towards heart failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the stress tolerance index for the area under risk.Pre and post-treatment mean values for Group I-T (VEGF) and Group I-P(placebo) are compared. The post-treatment value of Group I-T is higherto the pre-treatment value of the same group. The post-treatment valueof Group I-T is higher the post-treatment value of Group I-P.Intra-group paired comparisons show: 1) absence of statisticallysignificant differences between pre and post-treatment indexes for GroupI-P and 2) presence of statistically significant differences between preand post-treatment indexes for Group I-T. The non-paired comparisonsbetween groups show: 1) absence of statistically significant differencesbetween pre-treatment indexes for Group I-T and Group I-P and 2)presence of statistically significant differences between post-treatmentindexes for Group I-T and Group I-P.

FIG. 2 illustrates the perfusion improvement index for the area underrisk. Mean values for Group I-T (VEGF) and Group I-P (placebo) arecompared. The value for Group I-T is significantly higher than the valuefor Group I-P.

FIG. 3 shows the length density for the area under risk. Mean values forblood vessels with smooth muscle layer ranging from 8 to 50 μm areillustrated. The value for Group I-T (VEGF) is significantly higher thanthe value for Group I-P (placebo).

FIG. 4 shows the numerical density for the area under risk. Mean valuesfor blood vessels with smooth muscle layer ranging from 8 to 50 μm areillustrated. The value for Group I-T (VEGF) is significantly higher thanthe value for Group I-P (placebo).

FIG. 5 shows the length density for the area under risk. Mean values forblood vessels with smooth muscle layer ranging from 8 to 30 μm areillustrated. The value for Group I-T (VEGF) is significantly higher thanthe value for Group I-P (placebo).

FIG. 6 illustrates the effect of ischemia and treatment on cardiomyocyteKi67-positive nuclei and mitosis. FIG. 6A shows Ki67-positivecardiomyocyte nuclei index. No significant differences exist betweenGroup I-T (VEGF) and Group I-P (placebo) individuals. FIG. 6B showsGroup I-T individuals (VEGF) with a significantly higher cardiomyocytemitotic index for the area under risk (ischemic area) and thesurrounding myocardial tissue (non-ischemic area) as compared with GroupI-P individuals. The value for Group I-T (VEGF) is significantly higherthan the value for Group I-P (placebo).

FIG. 7 represents the VEGF mRNA transcription curve of the Group II-Tindividuals. The curve shows a peak by day 10 post-injection of thepUVEK15 plasmid.

FIG. 8 illustrates the metaphase of a cardiomyocyte from a Group I-Tindividual. Metaphasic cromosomes and mitotic spindle are clearlyvisible.

FIG. 9 illustrates the telophase of a cardiomyocyte from a Group I-Tindividual. Sarcomeric striations are clearly visible.

FIG. 10 illustrates the mitotic process of two adjacent cardiomyocytes.The boundary between the cardiomyocytes is distinguishable. Theintegrity of both cardiomyocytes is clearly observed.

FIG. 11 shows the non-conventional cytokinesis of a cardiomyocyte from aGroup I-T individual. Opposite chromosome plates in two adjacentcardiomyocytes are observed. The arrow indicates a possible splittinginto daughter cells. Bar=10 μm.

FIGS. 12 and 13 illustrate blood vessels with smooth muscle layer inmyocardial tissue of a Group I-T individual. Vascular smooth muscle wasidentified with alpha-actin immunohistochemical stain.

DESCRIPTION OF THE INVENTION

This invention relates, e.g., to a method for inducing arteriogenesis,lymphangiogenesis, vasculogenesis, or cardiomyogenesis, comprisingadministering to a cell or tissue in need thereof a dose of apolynucleotide that encodes a vascular endothelial growth factor (VEGF),or that encodes a polypeptide comprising an active site of the VEGF. Inthe method, the coding sequence is operably linked to an expressioncontrol sequence, and the dose is sufficient to induce arteriogenesis,lymphangiogenesis, vasculogenesis, or cardiomyogenesis. In a preferredembodiment, the VEGF is VEGF₁₋₁₆₅, whose amino acid sequence isrepresented by SEQ ID NO: 1.

The invention also relates to a method for inducing mitosis orproliferation of a smooth muscle cell, a skeletal muscle cell, or acardiomyocyte, comprising administering to a cell in need thereof a doseof a polynucleotide that encodes a vascular endothelial growth factor(VEGF), or that encodes a polypeptide comprising an active site of theVEGF. In this method, the coding sequence is operably linked to anexpression control sequence, and the dose is sufficient to induce themitosis or proliferation. In a preferred embodiment, the VEGF isVEGF₁₋₁₆₅, whose amino acid sequence is represented by SEQ ID NO: 1. Insome embodiments, the method is a method of tissue regeneration.

In preferred embodiments, the method of the invention is carried out invivo, and a sufficient dose of the polynucleotide is administered to asubject in need of such treatment to induce arteriogenesis,lymphangiogenesis, vasculogenesis, or cardiomyogenesis, and/or to inducethe mitosis or proliferation of a smooth muscle cell, a skeletal musclecell, or a cardiomyocyte.

The invention also relates to a method for inducing arteriogenesis,lymphangiogenesis, vasculogenesis, or cardiomyogenesis, comprisingadministering to a cell or tissue in need thereof a dose of a VEGFpolypeptide, or a polypeptide comprising an active site of the VEGF, thedose being sufficient to induce arteriogenesis, lymphangiogenesis,vasculogenesis, or cardiomyogenesis. The invention also relates to amethod for inducing mitosis or proliferation of a a smooth muscle cell,a skeletal muscle cell, or a cardiomyocyte, comprising administering tothe cell a sufficient dose of a VEGF polypeptide, or a polypeptide thatcomprises an active site of the VEGF, to induce the mitosis orproliferation. In preferred embodiments, the VEGF is VEGF₁₋₁₆₅, whoseamino acid sequence is represented by SEQ ID NO: 1.

The invention also relates to kits suitable for carrying out methods ofthe invention. In one embodiment, the kit comprises a polynucleotide orpolypeptide of the invention and a label or instructions indicating ause for the polynucleotide or polypeptide to induce arteriogenesis,lymphangiogenesis, vasculogenesis, cardiomyogenesis, or to inducemitosis or proliferation of a smooth muscle cell, a skeletal musclecell, or a cardiomyocyte. In another embodiment, the kit comprises adose of a polynucleotide or polypeptide of the invention that issufficient to induce mitosis or proliferation of a a smooth muscle cell,a skeletal muscle cell, or a cardiomyocyte, and/or to induce induce themitosis or proliferation of a smooth muscle cell, a skeletal musclecell, or a cardiomyocyte.

The terms myocardiogenesis and cardiomyogenesis are understood by thosein the art to be the same, and the terms are used interchangeablyherein.

One advantage of the present invention is the secure and efficientinduction of neovascular formation in hypoperfused and normoperfusedtissues. By utilizing embodiments of the invention, it is possible,e.g., to stimulate the neoformation, development, proliferation andgrowth of vessels. Embodiments of the invention are effective also forthe neoformation, development, proliferation and growth of smooth andstriated muscular cells. The method is particularly useful for inducingrevascularization in patients with ischemic heart disease. The presentinvention can also be used in the treatment of patients with peripheralartery disease or severe limb ischemia. The present invention can beutilized as sole therapy of ischemic diseases or associated withconventional revascularization procedures. The claimed method ischaracterized by the absence of adverse side effects related to thesystemic exposure to angiogenic factors in high doses.

Another advantage of the present invention is the regeneration ofmyocardial tissue (myocardiogenesis). The claimed method includes theinduction of cardiomyocyte mitosis and/or proliferation. In this way,the claimed method replaces infarcted tissue with autologous cardiacmuscle. The present invention reverts also the natural development ofhypertrophic and dilated cardiomyopathies of any etiology by inducingthe mitotic process in polyploid hypertrophic cardiomyocytes and byimproving tissue perfusion (i.e. inducing neovascular formation). Thiscircumstance results in higher number of normal daughter cells. Thesedaughter cells have a better perfusion compared to hypertrophic cells.All these advantages indicate that the present invention improves theshort, mid and long-term clinical and histophysiological outcomes ofheart disease.

Another potential advantage of this invention is its use in transplantmedicine. The claimed invention may be particularly useful intransplanted patients with chronic graft rejection and diffuse coronarydisease. The myocardial revascularization induced by the claimed methodwould restore the impaired perfusion and function in these patients.These patients are frequently not eligible for conventionalrevascularization methods (CABG, PTCA). The present invention representsan effective alternative revascularization strategy for these patients.

An additional potential advantage of the present invention is its usefor increasing perfusion in ischemic tissues of patients withdiabetes-related micro and macroangiopathy. The claimed method mayrevert or reduce chronic complications associated to diabetes such asdiabetic neuropathy, vasa-vasorum disease, ischemic heart disease,peripheral artery disease and severe limb ischemia, among others. SeeSchratzberger, et al., J. Clin. Invest., 107:1083-1092 (2001); Rivard,et al., Circulation 96 Suppl I: 175 (1997); Rivard, et al., Am. J.Pathol., 154: 355-363 (1999).

One of the advantages of the claimed method is its high safety when usedalong with minimally invasive procedures of percutaneousintramyocardial-transendocardial administration. This administration canbe achieved by accessing the left ventricular chamber through a cathetermediated endovascular approach. This type of administration may beassisted by fluoroscopy or an electromechanical mapping of the leftventricle. In this way the morbility and mortality associated toopen-chest surgery is significantly diminished.

In one embodiment of the invention, an inducing agent is administered toa cell, tissue, or subject in need thereof, wherein the inducing agentis a polynucleotide that encodes a VEGF and/or that encodes apolypeptide comprising an active site of the VEGF. As used herein, thesingular forms “a”, “an” and “the” include plural referents unless thecontext clearly dictates otherwise. For example, “an” active site, asused above, means one or more active sites. The VEGF polynucleotide mayencode a full-length VEGF polypeptide; or it may encode a polypeptideconsisting of one or more active sites of VEGF; or it may code apolypeptide consisting essentially of one or more active sites (e.g.,sequences of intermediate length, which contain amino acids in additionto those of the active site, wherein the additional amino acids do notaffect the basic and novel characteristics (e.g., activity) of theactive site). In preferred embodiments, the VEGF is VEGF₁₋₁₆₅, whoseamino acid sequence is represented by SEQ ID NO: 1.

A polynucleotide utilized according to the present invention may be,e.g., genomic DNA, cDNA or a messenger RNA. Preferably, thepolynucleotide is a cDNA.

In preferred embodiments, a coding sequence as above is operably linkedto an expression control sequence. As used herein, the term “expressioncontrol sequence” means a polynucleotide sequence that regulatesexpression of a polypeptide coded for by a polynucleotide to which it isfunctionally (“operably”) linked. Expression can be regulated at thelevel of the mRNA or polypeptide. Thus, the term expression controlsequence includes mRNA-related elements and protein-related elements.Such elements include promoters, domains within promoters, upstreamelements, enhancers, elements that confer tissue or cell specificity,response elements, ribosome binding sequences, transcriptionalterminators, etc. An expression control sequence is operably linked to anucleotide sequence (e.g., a coding sequence) when the expressioncontrol sequence is positioned in such a manner to effect or achieveexpression of the coding sequence. For example, when a promoter isoperably linked 5′ to a coding sequence, expression of the codingsequence is driven by the promoter. Suitable expression controlsequences will be evident to the skilled worker.

Expression control sequences which can be used in methods of theinvention, including both regulatable and constitutive controlsequences, are well-known to those of skill in the art. Preferredexpression control sequences are derived from highly-expressed genes,e.g., from genes encoding glycolytic enzymes such as 3-phosphoglyceratekinase (PGK), α-factor, acid phosphatase, or heat shock proteins, amongothers. Such expression control sequences can be selected from anydesired gene, e.g using CAT (chloramphenicol transferase) vectors orother vectors with selectable markers. Particular named eukaryoticpromoters include CMV immediate early, HSV thymidine kinase, early andlate SV40, adenovirus promoters, LTRs from retrovirus, and mousemetallothionein-1. Selection of an appropriate vector and exptressioncontrol sequence is well within the level of ordinary skill in the art.

In another embodiment of the invention, an inducing agent isadministered to a cell, tissue, or subject in need thereof, wherein theinducing agent is a VEGF polypeptide, and/or a polypeptide comprising anactive site of the VEGF. The VEGF may consist of, consist essentiallyof, or comprise, an active site of a VEGF. In a preferred embodiment,the VEGF is VEGF₁₋₁₆₅, whose amino acid sequence is represented by SEQID NO: 1.

According to the present invention, the inducing agent is administeredto a eukaryotic cell or a tissue composed of eukaryotic cells, such as amammalian cell or a tissue composed of mammalian cells. Preferably,mammalian cells are of porcine and human origin. More preferably, cellsare of human origin.

In one embodiment, the eukaryotic cells are muscle cells. Preferably,the muscle cells are cardiomyocytes, skeletal myoblasts, skeletalstriated muscle cells type I and type II, vascular smooth muscle cellsor non-vascular smooth muscle cells or myoepithelial cells. Morepreferably, the muscle cells are cardiomyocytes.

One embodiment of the invention is the induction of neovascularformation. Preferably, the induced neovascular formation is localized inthe site of administration of the inducing agent. More preferably, thesite of administration is the myocardium.

Another embodiment of the invention is the induction of localizedangiogenesis, either in vivo or ex vivo. Preferably, angiogenesis islocalized at the administration site of the inducing agent. Morepreferably, the site of administration is the myocardium. In anembodiment of the present invention, angiogenesis is induced innormoperfused tissue, either in vivo, in vitro or ex vivo. In anotherembodiment of the present invention, angiogenesis is induced in ischemictissue, either in vivo, in vitro or ex vivo. Preferably, theangiogenesis is induced in hypoperfused myocardial tissue, either invivo, in vitro or ex vivo. Hypoperfused myocardial tissue may beischemic, viable, hibernated, stunned, preconditioned, injured,infarcted, non-viable, fibrosed or necrosed. More preferably, theclaimed method induces angiogenesis in vivo in hypoperfused myocardialtissue.

Another embodiment of the invention is the induction of arteriogenesisin vivo, in vitro or ex vivo. Preferably, arteriogenesis is localized atthe site of administration. More preferably, the site of administrationis the myocardium. In an embodiment of the present invention,arteriogenesis is induced in normoperfused tissue in vivo, in vitro orex vivo. In another embodiment of the present invention, arteriogenesisis induced in ischemic tissue, in vivo, in vitro or ex vivo. Preferably,the arteriogenesis is induced in hypoperfused myocardial tissue in vivo,in vitro or ex vivo. Hypoperfused myocardial tissue may be ischemic,viable, hibernated, stunned, preconditioned, injured, infarcted,non-viable, fibrosed or necrosed. More preferably, the claimed methodinduces arteriogenesis in hypoperfused myocardial tissue in vivo.

Another embodiment of the invention is the induction of vasculogenesisin vivo, in vitro or ex vivo. Preferably, vasculogenesis is localized atthe site of administration. More preferably, the site of administrationis the myocardium. In an embodiment of the present invention,vasculogenesis is induced in normoperfused tissue in vivo, in vitro orex vivo. In another embodiment of the present invention, vasculogenesisis induced in ischemic tissue, in vivo, in vitro or ex vivo. Preferably,the vasculogenesis is induced in hypoperfused myocardial tissue, invivo, in vitro or ex vivo. Hypoperfused myocardial tissue may beischemic, viable, hibernated, stunned, preconditioned, injured,non-viable, infarcted, necrosed or fibrosed. More preferably, thevasculogenesis is induced in hypoperfused myocardial tissue in vivo.

Another embodiment of the invention is the induction oflymphangiogenesis in vivo, in vitro or ex vivo. Preferably,lymphangiogenesis is localized at the site of administration. Morepreferably, the site of administration is the myocardium. In anembodiment of the present invention, lymphangiogenesis is induced innormoperfused tissue, in vivo, in vitro or ex vivo. In anotherembodiment of the present invention, lymphangiogenesis is induced inischemic tissue, in vivo, in vitro or ex vivo. Preferably, thelymphangiogenesis is induced in hypoperfused myocardial tissue, in vivo,in vitro or ex vivo. Hypoperfused myocardial tissue may be ischemic,viable, hibernated, stunned, preconditioned, injured, non-viable,infarcted, necrosed or fibrosed. More preferably, the lymphangiogenesisis induced in hypoperfused myocardial tissue in vivo.

Another embodiment of the invention is the induction of mitosis in vivo,in vitro or ex vivo. Preferably, mitosis is induced locally at the siteof administration. More preferably, the site of administration is themyocardium. In an embodiment of the present invention, mitosis isinduced in normoperfused tissue, in vivo, in vitro or ex vivo. Inanother embodiment of the present invention, mitosis is induced inischemic tissue in vivo, in vitro or ex vivo. Preferably, the mitosis isinduced in hypoperfused myocardial tissue, in vivo, in vitro or ex vivo.Hypofused myocardial tissue may be ischemic, viable, hibernated,stunned, preconditioned, injured, non-viable, infarcted, necrosed orfibrosed. More preferably, the mitosis is induced in hypoperfusedmyocardial tissue in vivo.

The method also relates to the induction of proliferation of cells inwhich mitosis has been induced. In preferred embodiments, the mitosis orproliferation is in smooth muscle cells, skeletal muscle cells, orcardiomyocytes. In embodiments of the invention, a smooth muscle cell,skeletal muscle cell or cardiomyocyte in which mitosis or proliferationis induced is in myocardial tissue, skeletal tissue, or muscle tissue.Any type of muscle tissue may be regenerated by methods of theinvention.

Another embodiment of the invention is the induction of tissueregeneration in vivo, in vitro or ex vivo. Preferably, tissueregeneration is induced locally at the site of administration. Morepreferably, the site of administration is the myocardium. In anembodiment of the present invention, tissue regeneration is induced innormoperfused territories, in vivo, in vitro or ex vivo. In anotherembodiment of the present invention, tissue regeneration is induced inischemic territories, in vivo, in vitro or ex vivo. Preferably, thetissue regeneration is induced in hypoperfused myocardial territories,in vivo, in vitro or ex vivo. Hypoperfused myocardial territory may beischemic, viable, hibernated, stunned, preconditioned, injured,non-viable, infarcted, necrosed or fibrosed. More preferably, the tissueregeneration is induced in hypoperfused myocardial territories in vivo.

In one embodiment of the present invention, the coding nucleotidesequence is inserted in a vector. In embodiments of the claimed method,the vector is a viral vector such as adenovirus, adeno-associated virus,retrovirus or lentivirus. In another embodiment of the present method,the vector is a plasmid vector. More preferably, the coding sequenceinserted in a plasmid vector is pUVEK15.

Methods for inserting VEGF-coding sequences into vectors areconventional. Some suitable molecular biology methods, for use in theseand other aspects of the invention, are provided e.g., in Sambrook, etal. (1989), Molecular Cloning, a Laboratory Manual, Cold HarborLaboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al. (1995).Current Protocols in Molecular Biology, N.Y., John Wiley & Sons; Daviset al. (1986), Basic Methods in Molecular Biology, Elseveir SciencesPublishing, Inc., New York; Hames et al. (1985), Nucleic AcidHybridization, IL Press; Dracopoli et al. Current Protocols in HumanGenetics, John Wiley & Sons, Inc.; and Coligan et al. Current Protocolsin Protein Science, John Wiley & Sons, Inc.

In another embodiment of the present invention, the nucleotide sequence(e.g., when inserted into a plasmid vector) is transported by(administered to a cell or tissue by) a liposome. In an embodiment ofthe present invention, the inducing agent is in the form of apharmaceutical composition. The pharmaceutical composition isadministered to the recipient in sufficient doses.

A pharmaceutical composition used according to the present invention maybe administered by intravenous, intracoronary, intra-aortic,intrafemoral, intrapopliteal, intrapedialis, intra-posterior tibialis,intracarotideal and intraradialis routes. The pharmaceutical compoundmay be also administered by intrapericardial, intra-amniotic sac,intrapleural, intramyocardial-transepicardial,intramyocardial-transendocardial, intra-peripheral muscle, subcutaneous,intraspinal, and intracardiac (intra-atrial and intraventricular)routes. In addition, the inducing agent may be administered bysublingual, inhalatory, oral, rectal, periadventitial, perivascular,topical epicardial, topical epidermal, transdermal, ophthalmic routes orthrough the conjunctival, nasopharyngeal, bucopharyngeal,laryngopharyngeal, vaginal, colonic, urethral and vesical mucoses.Preferably, the inducing agent is administered byintramyocardial-transepicardial or intramyocardial-transendocardialinjections. More preferably, the inducing agent is administered byintramyocardial-transepicardial injection. In one embodiment ofparenteral administration, the polynucleotide is administered invehicles that are microbubbles, and the microbubbles are then disruptedby ultrasound directed at a site of interest, such that thepolynucleotide is released at and introduced into the site of interest.The ultrasound treatment permits one to direct the release of thepolynucleotide by disruption of the bubbles at the specific site atwhich the ultrasound is directed.

In an embodiment of the present invention, the inducing agent isinjected perpendicular to the plane of injection area. In anotherembodiment of the present invention, the inducing agent is injected inparallel to the plane of the area of injection. In another embodiment ofthe present invention, the inducing agent is injected in an obliqueangle in relation to the plane of the injection area. Preferably, theinducing agent is injected at an angle in relation to the plane of theinjection area of between about 30 degrees and about 90 degrees.Injections may be homogeneously or heterogeneously distributed in thearea of injection.

In a preferred embodiment, an inducing agent of the invention(polynucleotide or polypeptide) is formulated such that it isadministered under slow-release conditions. Any repeated administrationformulation or protocol may be used.

As used herein, “area of injection” includes the tissue territoryincluding the hypoperfused area, the transition area and normoperfusedarea surrounding the transition area. “Area of injection” may also bedefined as normal tissue.

As used herein, “area under risk” includes the myocardial area irrigatedby the circumflex coronary artery.

As used herein, “arteriogenesis” includes the formation, growth ordevelopment of blood vessels with a smooth muscle media layer.Angiogenesis (of any thin-walled vessel that does not contain smoothmuscle or a smooth muscle layer, e.g., a capillary vessel) is notencompassed by the term, arteriogenesis.

As used herein, “induce”, as well as the correlated term “induction”,refer to the action of generating, promoting, forming, regulating,activating, enhancing or accelerating a biological phenomenon. Anexample of induction is the action of VEGF as a vascular proliferationstimulator.

As used herein, “inducing agent” includes genomic DNA, cDNA or messengerRNA comprising sequences coding for the VEGF active site. “Inducingagent” also includes any vector containing a nucleotide sequence codingfor VEGF. “Inducing agent” is also defined as any polypeptide includingthe VEGF active site.

As used herein, “Ki67-positive cardiomyocyte nuclei index” refers to aparameter designed to assess the density of cycling (non-quiescent)cells in a tissue sample. This parameter refers to the number of Ki67positive cells per 10⁶ cardiomyocyte nuclei in an analyzed area.

As used herein, “length density index” refers to a parameter calculatedfor assessing a tissue vascularization. This parameter was designed toquantify vessels arranged in any variety of orientation. The method forcalculating this index is known in the art. See Anversa et al., Am. J.Physiol., 260: H1552-H1560 (1991); Adair et al., Am. J. Physiol., 266:H1434-H1438 (1994); Anversa et al., Am. J. Physiol., 267: H1062-H1073(1994).

As used herein, “localized” is a response restricted to the area ortissue of interest.

As used herein, “lymphangiogenesis” includes the formation, growth,development or proliferation of lymphatic vessels.

As used herein, “mammal” includes a warm blooded vertebrate animal whoseprogeny feeds with milk secreted by its mammary glands. The term“mammal” includes, but is not limited to, rats, mice, rabbits, dogs,cats, goats, sheep, cows, pigs, primates and humans.

As used herein, “mitosis” refers to the complete cell division process.

As used herein, “mitotic index” refers to a parameter designed to assessthe density of mitosis in a tissue sample. This parameter refers to thenumber of mitosis per 10⁶ cardiomyocyte nuclei in an analyzed area.

As used herein, “skeletal muscle cells” include striated muscle cells ofmuscle tissue and its precursors and progenitors, including skeletalmyoblasts and skeletal muscle satellite cells.

As used herein, “neovascular formation” includes the creation, growth,development or proliferation of blood vessels. Neovascular proliferationincludes arteriogenesis, vasculogenesis and lymphangiogenesis.

As used herein, “non-paired comparison” refers to the statisticalcomparison between two different groups of individuals at the same time.

As used herein, “paired comparison” refers to the statistical comparisonof the same group of individuals at different times.

As used herein, “perfusion improvement index” refers to a parameterdesigned to assess the overall improvement of left ventricularmyocardial perfusion. This index is calculated by the arithmeticaldifference between the post-treatment stress tolerance index and thepre-treatment stress tolerance index.

As used herein, a “pharmaceutical composition” of the inventioncomprises a polynucleotide or polypeptide of the invention and apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier can be, e.g., a solvent, adjuvant or excipient used toadminister an inducing agent. Pharmaceutical compositions can compriseany solvent, dispersion media, aqueous, gaseous solutions, antibacterialor antifungal agents, isotonic agents, either absorption delayer oraccelerator agents, or similar substances. The use of said substances inthe administration of pharmaceutically active compositions is known inthe art. Supplementary active ingredients may also be incorporated tothe pharmaceutical composition utilized in the present invention.Pharmaceutical compositions can comprise, but are not limited to, inertsolid fillings or solvents, sterile aqueous solutions and non-toxicorganic solvents. The pharmaceutically acceptable carrier should notreact with or reduce in any other manner the efficiency or stability ofthe inducing agent. Pharmaceutically acceptable carriers include, butare not limited to, water, ethanol, polyethileneglycol, mineral oil,petrolatum, propyleneglycol, lanolin and similar agents. Pharmaceuticalcompositions for injection include sterile aqueous solutions (whensoluble in water) or dispersions and sterile powders for extemporaneouspreparation of sterile dispersions or injectable solutions. In allcases, the formulation should be sterile. The formulation may be fluidto facilitate syringe dispensation. The formulation should also bestable under manufacturing and storage conditions and should bepreserved against the contaminant action of microorganisms such asbacteria, viruses and fungi.

As used herein, “post-treatment stress tolerance index” refers to aparameter designed to assess the left ventricular myocardial perfusionin post-treatment conditions. This index is calculated by thearithmetical difference between the post-treatment percentual perfusionvalue during pharmacological challenge (stress) and the post-treatmentpercentual perfusion value at rest.

As used herein, “pre-treatment stress tolerance index” refers to aparameter designed to assess the left ventricular myocardial perfusionin pre-treatment conditions. This index is calculated by thearithmetical difference between the pre-treatment percentual perfusionvalue during pharmacological challenge (stress) and the pre-treatmentpercentual perfusion value at rest.

As used herein, “stress tolerance index” is defined as the arithmeticaldifference between the percentual perfusion value during pharmacologicalchallenge (stress) and the percentual perfusion value at rest. Thisindex is calculated in post-treatment and pre-treatment situations.

In preferred embodiments, a method of the invention is carried out invivo. A sufficient dose of an inducing agent (e.g., a polynucleotide orpolypeptide of the invention) is administered to a subject (e.g., apatient) in need of such treatment. A subject “in need of suchtreatment” can be, e.g., a subject who exhibits signs or symptoms of, orwho is suffering from, one of the mentioned conditions. “Signs” of acondition are manifestations assessed by physical examination, EKG orother methods, which are not full-fledged symptoms, but which arerecognizable by a physician. For example, the subject may exhibit signsor symptoms of, or may suffer from, myocardial infarction, myocardialischemia, dilated cardiomyopathy, or hypertrophic cardiomyopathy.Preferably, the subject is a human patient.

As used herein, a “sufficient dose” is a quantity of the inducing agent,or of a pharmaceutical composition including the inducing agent, whichis adequate to attain at least a detectable amount of the specifiedfunction. In the context of the present invention, “sufficient dose”refers to a quantity of the inducing agent, or of the pharmaceuticalcomposition including the inducing agent, which is adequate to produce,e.g., one or more of the following results: 1) the induction ofarteriogenesis, vasculogenesis, lymphangiogenesis, or myocardiogenesisin eukaryotic cells, 2) the activation of the cell cycle in eukaryoticcells, 3) the induction or acceleration of the mitotic process ineukaryotic cells, e.g. the induction of mitosis or proliferation of asmooth muscle cell, a skeletal muscle cell, or a cardiomyocyte.

The sufficient dose for any particular use will vary from subject tosubject, depending on, i.a., the species, age, weight and general orclinical condition of the subject, the severity or mechanism of anydisorder being treated, the particular agent or vehicle used, the methodand scheduling of administration, and the like. A therapeuticallysufficient dose can be determined empirically, by conventionalprocedures known to those of skill in the art. See, e.g., ThePharmacological Basis of Therapeutics, Goodman and Gilman, eds.,Macmillan Publishing Co., New York. For example, a sufficient dose canbe estimated initially either in cell culture assays or in suitableanimal models. The animal model may also be used to determine theappropriate concentration ranges and routes of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. For example, the doses administered to pigs inthe Examples herein can be converted to suitable doses for humans. Asufficient dose can also be selected by analogy to doses for comparabletherapeutic agents.

In general, sufficient doses of VEGF-encoding polynucleotides vary frombetween about 0.003 to about 0.36 nmoles/kg body weight, depending onthe route of administration and other factors as noted above. In apreferred embodiment, the dose is between about 0.01 and about 0.10nmoles/kg. The nmoles are of polynucleotide encoding an active VEGFpolypeptide. As used herein, an “active VEGF polypeptide” is apolypeptide that comprises an active site of a VEGF polypeptide, e.g.,full-length VEGF or an active site thereof. The dose may be administeredas a single dose, or in multiple doses (e.g., two or more doses) over anempirically determined amount of time. For example, the dose may beadministered in two or more events, at different times, such as two ormore weeks apart.

For polynucleotides in adenoviral viral vectors, a sufficient dose mayvary between about 2.5×10¹⁰ and about 10×10¹⁵ pfu (plaque formingunits), more preferably between about 3×10¹⁰ and about 10×10¹² pfu.Comparable doses for other viral vectors will be evident to the skilledworker.

In general, it is preferable to formulate a polynucleotide of theinvention in as concentrated a solution as possible. For example, in oneembodiment of the invention, in which the polynucleotide coding sequenceis inserted in a vector to form the plasmid, pUVEK15^(VEGF) aconcentration of between about 0.5 to about 4 mg/mL of the plasmid ispreferred. Comparable concentrations of other vectors containing thecoding sequences, or of polypeptides, will be evident to the skilledworker.

In general, sufficient doses of VEGF polypeptides vary from betweenabout 0.35 and about 3.5 mg/kg body weight, depending on the route ofadministration and other factors as noted above. In a preferredembodiment, the dose is between about 0.4 and about 1.4 mg/kg. Themgrams are of active VEGF polypeptide. The dose may be administered as asingle dose, or in multiple doses (e.g., two or more doses) over anempirically determined amount of time.

As used herein, “vasculogenesis” includes the formation, growth,development or proliferation of blood vessels derived fromundifferentiated or underdifferentiated cells.

As used herein, “VEGF” includes any vascular endothelial growth factor.“VEGF” includes, but is not limited to, the VEGF variants A, B, C, D, Eand F. See Hamawy, et al., Curr. Opin. Cardiol., 14:515-522 (1999);Neufeld, et al., Prog. Growth Factor Res., 5:89-97 (1994); Olofsson, etal., Proc. Natl. Acad. Sci. USA, 93:2576-2581 (1996); Chilov, et al., J.Biol. Chem., 272:25176-25183 (1997); Olofsson, et al., Curr. Opin.Biotechnol., 10:528-535 (1999). The VEGF A variant includes, but is notlimited to, isoforms VEGF₁₋₁₂₁, VEGF₁₋₁₄₅, VEGF₁₋₁₆₇, VEGF₁₋₁₆₅,VEGF₁₋₁₈₉ and VEGF₁₋₂₀₆. The SEQ ID NO. 1 illustrates an example ofisoform VEGF₁₋₁₆₅. See Tischer, et al., J. Biol. Chem., 266:11947-11954(1991); Poltorak, et al., J. Biol. Chem., 272:7151-7158 (1997). The term“VEGF” also includes the vascular permeability factor or vasculotropin(VPF). See Keck, et al., Science 246:1309-1312 (1989); Senger, et al.,Science, 219:983-985 (1983). VPF is currently known in the art as VEGFA. Other members of the VEGF family can also be used, includingplacental growth factors P1GF I and II.

The sequences of suitable VEGFs are readily available, e.g., on the website of the National Center for Biotechnology Information (NCBI). Forexample, the loci for human VEGF family members include: VEGF-A-P15692and NP003367; VEGF-B-NP003368, P49765, AAL79001, AAL79000, AAC50721,AAB06274, and AAH08818; VEGF-C-NP005420, P49767, 569207, AAB36425, andCAA63907; VEGF-D-NP004460, AAH27948, 043915, CAA03942 and BAA24264;VEGF-E-AAQ88857; VEGF-F-2VPFF; P1GF-1-NP002623, AAH07789, AAH07255,AAH01422, P49763, CAA38698 and CAA70463; synthetic constructs of ChainA-1FZVA and Chain B-1FZVB of P1GF-1; and P1GF-2-AAB25832 and AAB30462.

In preferred embodiments, the VEGF is of human origin. However, VEGFfrom other species, such as mouse, may also be used.

Structure/function analysis has identified a number of sequences andamino acid residues of VEGF that are important for its activity. Thus,it would be evident to a skilled worker which residues constitute an“active site” for any particular VEGF activity. A review of some of thestructure/function studies follows:

In the 1980s VEGF was identified independently as vascular permeabilityfactor (VPF) and as vascular endothelial cell-specific growth factor(Senger 1983, Leung 1989). Molecular cloning of the genes encoding these“two” proteins clarified that they are essentially the same proteinencoded by a single gene (VEGF gene). Therefore, this protein isreferred as VEGF, VEGF/VPF or, sometimes, as VPF.

X-ray crystallography of a VEGF fragment (residues 8-109) showed thatVEGF belongs to the dimeric cysteine-knot growth factor superfamily(Muller 1997). Each monomer is characterized by an intra-chaindisulphide bonded knot motif at one end of a four-stranded β sheet (McDonald 1993, Murray-Rust 1993, Sun 1995). One subdivision of thissuper-family is the PDGF (platelet-derived growth factor) gene family,to which VEGF belongs. Among these gene products, 8 cysteine residuesare conserved at the same positions, and these products function as adimer form, since 2 out of 8 cysteines generate intermolecularcross-linking (S—S bonds motif). The other 6 cysteines make 3intramolecular S—S bonds to form 3 loop structures (Wiesmann 1997). Themonomers are held in a “side-by-side” orientation, the two β sheetslying perpendicular to the twofold-symmetry axis. The structure of theVEGF165 heparin-binding region (residues 111-165) has been solvedseparately by NMR and represents a novel type of heparin-binding domain(Fairbrother 1998).

All VEGF isoforms are secreted as covalently linked homodimers. Monomersassociate initially through hydrophobic interactions and are thenstabilized by disulphide bonding between Cys51 of one chain and Cys61 ofthe other (Potgens 1994). The signal peptide (exon 1 and four residuesof exon 2), which includes an amphipathic α-helix (residues 12-19)essential for this dimerization, is cleaved off during secretion (Leung1989, Keck 1989, Siemeister 1998a). A potential N-glycosylation siteexists at Asn74 and apparently has no effect on VEGF function but isrequired for efficient secretion (Peretz 1992, Claffey 1995). And it isimportant to remark that the secretion process is necessary for at leastsome of the VEGF biological activities (that depend on VEGF binding toother cells receptors).

Potgens et al. showed that covalent dimerization of VEGF is essentialfor effective receptor binding and biological activity (Potgens 1994).They found that VEGF mutants lacking cysteine residue 2 or 4 (directlyinvolved in anti-parallel inter-chain disulphide bonds) competed poorlyfor receptor binding of labeled VEGF and had low biological activity,thus VEGF needs to be a covalent dimer for efficient receptor bindingand activation. Furthermore, they also found that cysteine residue 5 wasessential for VEGF dimerization and activity, while the mutant lackingcysteine residue 3 was only mildly affected in its ability to dimerizeand had partial biological activity (Potgens 1994).

Alanine-scanning analysis was performed to identify a positively chargedsurface in VEGF that mediates receptor binding (Ferrara 1997).Site-directed mutagenesis identified three acidic residues (Asp63, Glu64and Glu67) in exon 3, and three basic residues (Arg82, Lys84 and His86)in exon 4 that are essential for binding to VEGF receptors VEGFR-1 andVEGFR-2, respectively. The most significant effect on endothelial cellproliferation was observed with mutations in the 82-86 region (Ferrara1997, Key 1996a). Three highly flexible loops are clustered at each poleof VEGF at the dimer interface. Loop II contains the VEGFR-1 bindingdeterminants and lies close to loop III of the opposing monomer, whichbinds to VEGFR-2 (Keyt 1996a). The positioning of these receptor-bindinginterfaces at each pole of VEGF seems to facilitate receptordimerization, which is essential for transphosphorylation andsignalling, because mutant dimers that have only one receptor-bindingsite antagonize native VEGF activity (Siemeister 1998b).

The binding sites to extracellular matrix (ECM) seem to be alsoimportant for VEGF action. VEGF isoforms in the ECM constitute areservoir of growth factor that can be slowly released by exposure toheparin, heparan sulphate and heparinases or more rapidly mobilized byspecific proteolytic enzymes such as plasmin and urokinase-typeplasminogen activator uPA (Houck 1992, Plouet 1997). These enzymesalready contribute to vascular proliferation through ECMdepolymerization and, as well as releasing sequestered VEGF from thecell surface and ECM, might also regulate VEGF bioactivity. Keyt et al.found that the removal of the carboxyl-terminal domain of VEGF165 isassociated with a significant loss in bioactivity (Keyt 1996b).

Other relevant issues are the VEGF mediated synthesis or secretion ofother growth factors or VEGF interaction with different mitogens toachieve the biological effects. For example VEGF has been shown toupregulate PDGF-BB (Hirschi 1998). Other example is the sequence encodedby exon 6 (not present in VEGF165) has also been shown to releasebioactive bFGF from the ECM and cell surface and thus confers theability to exert some of VEGF biological effects through bFGF signallingpathways (Jonca 1997)

A VEGF polypeptide used in methods of the invention may be a fragment orvariant of a naturally occurring VEGF polypeptide, provided that thefragment or variant retains an activity of the naturally occurringpolypeptide which allows it to achieve a result of a method of theinvention. Such a fragment or variant is referred to herein as an“active fragment” or “active variant.”

An active fragment of a VEGF polypeptide may be of any size that iscompatible with the invention, e.g., a polypeptide that is shorter thana naturally occurring VEGF, but that retains an active site of the VEGF.

An active variant of a VEGF polypeptide may be, e.g., (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue), which substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which additional amino acids are fused to thepolypeptide, such as a leader or secretory sequence or a sequence whichis employed for purification of the polypeptide, commonly for thepurpose of creating a genetically engineered form of the protein that issusceptible to secretion from a cell, such as a transformed cell. Theadditional amino acids may be from a heterologous source, or may beendogenous to the natural gene. Examples of all of these types ofvariants will be evident to a skilled worker. Among the preferredmodifications are glycosylation or PEGylation of the protein, and/oramino acid substitutions, which increase bioavailaility, biologicalactivity, biological effect, and/or half-life of the protein.

The invention also encompasses active fragments or variants of naturallyoccurring polynucleotides encoding VEGF. Such an active fragment orvariant retains an activity of the naturally occurring polynucleotidewhich allows it to achieve a result of a method of the invention.Suitable variant polynucleotides include polynucleotides that encode anyof the fragments or variant polypeptides noted above. Also included arevariants which reflect the degeneracy of the genetic code, or which arenaturally occurring or artificially generated allelic variants of a wildtype polynucleotide.

Active variant polynucleotides of the invention may take a variety offorms, including, e.g., naturally or non-naturally occurringpolymorphisms, including single nucleotide polymorphisms (SNPs), allelicvariants, and mutants. They may comprise, e.g., one or more additions,insertions, deletions, substitutions, transitions, transversions,inversions, chromosomal translocations, variants resulting fromalternative splicing events, or the like, or any combinations thereof.

Other types of active variants will be evident to one of skill in theart. For example, the nucleotides of a polynucleotide can be joined viavarious known linkages, e.g., ester, sulfamate, sulfamide,phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc.,depending on the desired purpose, e.g., improved in vivo stability, etc.See, e.g., U.S. Pat. No. 5,378,825. Any desired nucleotide or nucleotideanalog can be incorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, etc.

Active variant polynucleotides or polypeptides of the invention includepolynucleotides or polypeptides having sequences that exhibit a percentidentity to one of the sequences noted above of at least about 70%,preferably at least about 80%, more preferably at least about 90% or95%, or 98%, provided that the polypeptide or polypeptide exhibits thedesired function noted above.

Methods of determining the degree of identity of two sequences areconventional. The comparison of sequences and determination of percentidentity and similarity between two sequences can be accomplished usinga mathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). Among suitable mathematical algorithms that can be usedare those described in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA90:5873-5877; those of the GAP program I the GCG software package(Devereux et al. (1984) Nucleic Acids Res. 12 (1):387); and thealgorithm of Myers and Miller, CABIOS (1989).

Alternatively, a suitable variant polynucleotide is one that hybridizesunder standard conditions of high stringency to a naturally occurringVEGF-encoding polynucleotide.

As used herein, “underdifferentiated cells” are cells with acharacteristic phenotypic profile but with the capacity of originatingcells with a different phenotypic profile. “Underdifferentiated cells”include, but are not limited to, fibroblasts, myoblasts, osteoblasts,endothelial precursor cells, skeletal muscle satellite cells, neuraltissue glial cells, stem cells, cardiac progenitor cells, and cardiacprecursor cells.

In one embodiment, the present invention employs a plasmid calledpUVEK15 of approximately 3086 base pairs (bp). The pUVEK15 plasmid ischaracterized by including a cytomegalovirus (CMV) promoter, a chimericintron, a DNA fragment containing a vascular endothelial growth factor(VEGF)-encoding sequence and a DNA sequence of approximately 1290 bp,which confers resistance to kanamicyn. The VEGF nucleotide sequencepresent in the pUVEK15 plasmid encodes the human 165 amino acid VEGFpolypeptide represented by SEQ ID NO: 1. The pUVEK15 plasmid isdeposited under the access number DSM 13833 at DSMZ—Deutsche Sammlungvon Mikroorganismen and Zellkulturen, Federal Republic of Germany.

Another embodiment of the invention is a kit suitable for carrying out amethod of the invention. For example, the kit may comprise (a) apolynucleotide that encodes a vascular endothelial growth factor (VEGF),or that encodes a polypeptide comprising an active site of the VEGF,wherein the coding sequence is operably linked to an expression controlsequence, and (b) a label or instructions indicating a use for thepolynucleotide to induce arteriogenesis, lymphangiogenesis,vasculogenesis, myocardiogenesis, or mitosis or proliferation of asmooth muscle cell, a skeletal muscle cell, or a cardiomyocyte.

In another embodiment, the kit comprises a dose of a polynucleotide thatencodes a vascular endothelial growth factor (VEGF), or that encodes apolypeptide encoding an active site of the VEGF, wherein the codingsequence is operably linked to an expression control sequence, the dosebeing sufficient to induce arteriogenesis, lymphangiogenesis,vasculogenesis, myocardiogenesis, or mitosis or proliferation of asmooth muscle cell, a skeletal muscle cell, or a cardiomyocyte.

In another embodiment, the kit comprises (a) a VEGF polypeptide, or apolypeptide the comprises an active site of the VEGF, and (b) a label orinstructions indicating a use for the polypeptide to inducearteriogenesis, lymphangiogenesis, vasculogenesis, or myocardiogenesis,or mitosis or proliferation of a smooth muscle cell, a skeletal musclecell, or a cardiomyocyte.

In another embodiment, the kit comprises a dose of a VEGf polypeptide,or a polypeptide that comprises an active site of the VEGF, the dosebeing sufficient to induce arteriogenesis, lymphangiogenesis,vasculogenesis, myocardiogenesis, or mitosis or to induce proliferationof a smooth muscle cell, a skeletal muscle cell, or a cardiomyocyte.

The reagents of a kit of the invention may be in containers in which thereagents are stable, e.g., in lyophilized form or stabilized liquids.The reagents may also be in single use form, e.g., in single dosageform.

Having described the invention in general terms, it will be more easilyunderstood by reference to the following examples which are presented asan illustration and are not intended to limit the present invention,save when specifically indicated.

EXAMPLES Example I Induction of Ischemia

Eighty Landrace pigs weighing approximately 25 kg (approx. 3 months ofage) were submitted to the following protocol: 1) each individualunderwent clinical and laboratory assessment of good health; 2) asterile thoracotomy was performed at the 4^(th) left intercostal spaceunder general anesthesia (induction: thiopental sodium 20 mg/kg;maintenance: 2% enflurane) and the circumflex coronary artery wasdissected free from surrounding tissue at its proximal portion; 3) anAmeroid constrictor was placed embracing the origin of the circumflexcoronary artery; and 4) the thoracotomy was repaired.

Example II Basal Pre-Treatment Studies

Three weeks after the first surgery indicated in the previous example,basal (pre-treatment) studies were performed on the individuals. Thestudies were conducted under sedation with sufficient doses ofintravenous sodium thiopental and under electrocardiographic control.Basal myocardial perfusion studies were performed on each individual.The left ventricular perfusion was quantified by single photon emissioncomputed tomography (SPECT) utilizing an ADAC Vertex Dual DetectorCamera System (ADAC Healthcare Information Systems Inc., USA). Sestamibimarked with ^(99m)Tc was utilized as contrast.

The studies were performed at rest and under pharmacological challengewith progressive doses of intravenous dobutamine. The dobutamineinfusion was interrupted when heart rate was at least a 50% above thebasal (rest) values.

Individuals fulfilling the inclusion criterium (hipoperfusion in aterritory consistent with the circumflex coronary artery bed) wereselected. Of the subjects considered, twenty six individuals developedchronic myocardial ischemia and were selected as satisfying theinclusion criterium.

Example III Administration of VEGF Plasmid and Placebo Plasmid

The twenty six individuals of the previous example were distributed intwo groups: A first group consisting of 16 individuals (Group I) and asecond group consisting of 10 individuals (Group II). Group Iindividuals were utilized to perform histopathological and physiologicalstudies. Group II individuals were utilized to assess the presence andexpression of the VEGF plasmid.

Group I individuals were randomized into two subgroups (Group I-T andGroup I-P) with the same number of members (4 females and 4 males persubgroup). The treated group was designated Group I-T. The placebo groupwas designated Group I-P.

Group II individuals were randomized into two subgroups (Group II-T andGroup II-P). Eight individuals were allocated to Group II-T. Twoindividuals were allocated to Group II-P. The treated group wasdesignated Group II-T. The placebo group was designated Group II-P.

A sterile reopening of the previous thoracotomy was performed eachindividual of both Group I and Group II (reoperation) under generalanesthesia (induction: sodium thiopental 20 mg/kg, maintenance: 2%enflurane).

Each individual from Groups I-T and II-T received 10 injections of asolution containing pUVEK15 plasmid encoding for vascular endothelialgrowth factor (1.9 mg of pUVEK15 in 1 mL of saline). Each injectioncontained 200 μl of the plasmid solution. Each individual received atotal dose of 3.8 mg of the pUVEK15 plasmid. Each individual from GroupsI-P and II-P received 10 injections of a solution containingpUVEK15^(−VEGF) plasmid without the encoding region for the vascularendothelial growth factor (1.9 mg of pUVEK15^(−VEGF) in 1 mL of saline).Each injection contained 200 μl of the plasmid solution. Each individualreceived a total dose of 3.8 mg of the pUVEK15^(−VEGF) plasmid.

Each aliquot was injected intramyocardically, starting from thenormoperfused left anterior descending artery territory (2-3 aliquots)and spanning the basal and mid zones of the anterolateral leftventricular wall. The area of injection included the hypoperfused zone,the transition zone and the normoperfused tissue immediately surroundingthe transition zone. The injections were administered at a 45 degreeangle in relation to the plane of the myocardium area, avoidingintraventricular administration of the solution. The injections werehomogeneously distributed in the area of injection. The thoracotomy wasrepaired in each individual after administration.

Example IV Post-Treatment Studies

1. Histopathological and Physiological Studies

Five weeks after the second surgery (reoperation), post-treatmentstudies were performed on Group I individuals. The individual weresedated with sufficient doses of intravenous sodium thiopental. The leftventricular perfusion was assessed for each individual following theprotocol described in example 2.

The individuals were euthanized using an overdose of thiopental sodiumfollowed by a lethal injection of potassium chloride. The heart,kidneys, liver, lungs, skeletal muscle, eyes and gonads were excised forhistopathological assessment, including neoangiogenesis and mitosisdeterminations. The histopathological studies were performed inmyocardial and peripheral tissues according to the following protocols.

For myocardial studies, the pericardium, adherent fat, atria and rightventricular free wall were removed. In each animal, the left circumflexcoronary artery was examined at the site of the Ameroid to assess forocclusion. Subsequently, the left ventricle, including the septum, wascut transversally at one third of the distance between the apex and themitral annulus. Subsequently, a slice of 5 mm in thickness was cut fromthe distal end of the upper third, rinsed in Ringer solution and fixedflat for 48 hours in 10% formaldehyde buffered solution. This slice waschosen in order to: 1) limit the analysis to areas clearly perfused byonly one vessel (left anterior descending coronary artery, leftcircumflex coronary artery or right coronary artery), without mixedsupply from more than one artery, and 2) match the histology with theperfusion data.

After fixation, the slice was divided into 6 blocks, corresponding, from1 to 6 to: the posterior half of the septum, the posterior wall, theposterolateral wall, the lateral wall, the anterior wall and theanterior half of the septum. These 6 blocks were embedded in Histowax™,and sections of 5 μm thickness were mounted on slides previously wettedin a 0.01% polylysine aqueous solution (Sigma Chemical Co., U.S.A.) anddried at 37° C. The sections were stained with hematoxylin-eosin.Identification of intramyocardial vessels was made under opticalmicroscopy. The endothelium was identified by immunohistochemistryemploying the biotin streptavidin technique and a monoconal antibodyagainst von Willebrand factor. The smooth muscle layer was identified byimmunohistochemistry to assess arteriogenesis. A monoclonal antibodyagainst alpha-actin (Biogenex Labs. Inc., U.S.A.) was utilized to thispurpose.

For quantitative analysis of collateral circulation a digital analysissystem was employed (Vidas Kontron, Germany). The analysis focused onarteriole-sized vessels (ranging from 8 to 50 μm of maximum diameter)with smooth muscle layer. The morphometric study was performed on thetotal slice area. The numerical and length density of collateral vesselswere determined. The numerical density was calculated as number ofcollaterals (n) per square milimeter (n/mm²) The collateral lengthdensity (Lc) was calculated with the methodology known in the art forvessels arranged in any variety of orientation. See Anversa et al., Am.J. Physiol., 260: H1552-H1560 (1991); Adair et al., Am. J Physiol., 266:H1434-H1438 (1994); Anversa et al., Am. J. Physiol., 267: H1062-H1073(1994). For n vessels encountered in an area A, Lc, expressed inmillimeters per unit volume of myocardium (mm/mm³), is equal to the sumof the ratio R of the long to the short axis of each vessel.

${Lc} = {{{1/A}{\sum\limits_{i = 1}^{n}R_{i}}} = {\left( {R_{1} + R_{2} + R_{3} + {\ldots \mspace{14mu} R_{n}}} \right)/A}}$

In addition, the length density for intramyocardial vessels ranging from8 to 30 μm of maximum diameter was also analyzed.

Both indexes (numerical and length density) were averaged for both theischemic (posterolateral, lateral, and anterolateral walls) and thenon-ischemic (septum, anterior and posterior walls) zones.

To evidence cardiomyocytes undergoing cell cycle and mitosis, two doubleimmunohistochemical techniques were used in the tissue sections of theGroup I individuals. The following protocols were performed:

-   -   (a) Tissue sections were incubated with a monoclonal antibody        against the Ki67 antigen (Novocastra Labs., U.K.). The Ki67 is a        protein expressed exclusively during the cell cycle which        identifies nuclei undergoing the G1, S and G2-M phases and        decorates condensed mitotic chromosomes. The Ki67 expression        pattern is not affected by DNA damage or by apoptosis. See Brown        et al., Histopathology, 17:489-503 (1990); Gerdes et al., J.        Immunol., 133:1710-1715 (1984); Ross et al., J. Clin. Pathol.,        48:M113-117 (1995). Subsequently, the sections were post-treated        with a biotinilated anti-mouse immunoglobulin antiserum        (Biogenex, USA), followed by peroxidase-labeled avidin and        revealed with AEC as chromogen. Afterwards, the sections were        incubated with an anti-sarcomeric α-actin antibody (Dako, USA)        to identify striated muscular cells. Subsequently, the sections        were post-treated with the biotinilated antiserum followed by        alkaline phosphatase-labeled streptavidin (Biogenex, USA) and        Fast Red as chromogen.    -   (b) Tissue sections were incubated with a monoclonal antibody        against the Ki67 antigen (Novocastra Labs., U.K.). The sections        were post-treated with biotinilated antibodies, and revealed        with fluorescein-labeled streptavidin (Vector, USA). Afterwards,        the sections were incubated with rhodamine-labeled phalloidin        (Sigma, USA), a protein binding F-actin, in order to identify        striated muscular cells.

The tissue sections treated with enzyme-labeled avidin were examinedwith light microscopy with Nomarski optics. The tissue sections stainedwith fluorescent reactants were examined with confocal microscopy(Zeiss, Federal Republic of Germany).

Cardiomyocyte nuclei (CMN) density (CMN per mm²) was determined bycounting the number of CMN in longitudinally oriented cells containingsarcomeric α-actin in a 5 mm² area of the lateral wall mesocardium. Thenumber of Ki67-positive CMN and the number of cardiomyocyte mitosis weredetermined in the whole ventricular tissue section area of eachindividual (total scanned area, TSA). The TSA of the Group I individualsaveraged 1345.7±289.7 mm².

The Ki67-positive CMN index was calculated as: [Ki67-positivenuclei/(TSA×CMN density)]×10⁶. The mitotic index was calculated as:[mitosis/(TSA×CMN density)]×10⁶. Data was expressed as number ofKi67-positive nuclei and number of cardiomyocyte mitosis per 10⁶ CMN.Both indexes were averaged for both the ischemic (posterolateral,lateral, and anterolateral walls) and the non-ischemic (septum, anteriorand posterior walls) zones for each individual.

For peripheral studies, the tissues were fixed in 10% formaldehydebuffered solution, sectioned in blocks and included in Histowax™paraffin. Tissue slices of 5 μm thickness were obtained from the blocksand stained with hematoxylin-eosin. An histopathological assessment forpossible toxic effects in remote tissues was made by optical microscopy.

2. Presence and Transcription of VEGF Plasmid in Myocardial Tissue

After the second surgery (reoperation) the Group II individuals wereeuthanized using an overdose of thiopental sodium followed by a lethalinjection of potassium chloride, according to the following chronogram:2 individuals from Group II-T after 3 days of reoperation, 2 individualsfrom Group II-T and 2 individuals from Group II-P after 10 days ofreoperation, 2 individuals of Group II-T after 16 days of reoperationand 2 individuals from Group II-T after 35 days of reoperation.Necropsies were performed in each euthanized individual. Myocardialtissue of the area under risk was obtained from each individual.

The molecular assessment was performed to detect the presence ofplasmidic DNA and its transcript (mRNA). The presence of plasmidic DNAand mRNA were determined by the polymerase chain reaction (PCR) and thereverse transcriptase-polymerase chain reaction (RT-PCR) techniques,respectively. See Mullis, et al., Meth. Enzymol., 55:335-350 (1987);Belyaysky, et al., Nucleic. Acids Res., 17:2919-2932 (1989).

Total RNA was isolated from tissue samples using Trizol reagent (GibcoBRL Life Technologies, USA) and treated with DNAse I (Promega, USA). RNAwas quantified by spectrophotometry at A_(260/280) nm. One μg of totalRNA was reverse transcripted using random hexamers (PerkinElmer, USA).Human VEGF was then amplified from cDNA using Taq polymerase(PerkinElmer, USA) with the oligonucleotide primers5′CAACATCACCATGCAGATT3′ and 5′GCAGGAATTCATCGATTCA3′ at cyclingconditions of 95° C. for 15 sec, 52° C. for 30 sec and 65° C. for 30sec, for 35 cycles. Non-competitive amplification of constitutive GAPDHwas used to demonstrate the presence of intact mRNA in each total RNAsample. RT-PCR was performed in myocardial tissue of Group II-Tindividuals without reverse transcriptase to assess the possiblecontamination with plasmidic DNA or genomic DNA. The results of thiscontrol reaction were negative, excluding the possibility ofcontamination.

Example V Results

1. Histopathological and Physiological Analysis The perfusion andhistopathological studies showed vascular formation and growth inmyocardial tissue of treated individuals. The histopathological studyalso revealed the induction of mitosis in cardiomyocytes, endothelialcells and smooth muscle cells of Group I-T individuals.

The stress tolerance index and perfusion improvement index weredetermined for each myocardial segment of all Group I individuals inorder to assess left ventricular perfusion. Mean values of both indexeswere calculated for the area under risk and the surrounding tissue foreach individual. Finally, the mean values for each group werecalculated.

The analysis of the perfusion in the area under risk revealed that:

-   -   (a) Group I-P: absence of statistically significant differences        between the pre-treatment and post-treatment stress tolerance        indexes (intra-group paired comparison). This result indicates        that the perfusion and stress tolerance did not improve in the        Group I-P individuals after the placebo treatment.    -   (b) Group I-T: presence of statistically significant differences        between the pre-treatment and post-treatment stress tolerance        indexes (intra-group paired comparison). The post-treatment mean        value was significantly higher than the pre-treatment mean        value. This result indicates that the perfusion and stress        tolerance improved significantly in the Group I-T individuals        after pUVEK15 treatment.    -   (c) Pre-treatment stress tolerance indexes: absence of        statistically significant differences between the pre-treatment        mean values of Group I-T individuals and Group I-P individuals        (inter-group non-paired comparison). This result demonstrates        that perfusion was homogenous for both subgroups before        treatment.    -   (d) Post-treatment stress tolerance indexes: presence of        statistically significant differences between the post-treatment        mean values of Group I-T individuals and Group I-P individuals        (inter-group non-paired comparison). The post-treatment mean        value of Group I-T was significantly higher than the        post-treatment mean value of Group I-P. This result indicates        that the perfusion and stress tolerance of Group I-T individuals        were higher than the Group I-P individuals after treatment with        pUVEK15.    -   (e) Perfusion improvement indexes: presence of statistically        significant differences between both subgroups. The mean value        for Group I-T individuals was significantly higher than the mean        value for Group I-P individuals (inter-group non-paired        comparison). This result indicates that the perfusion of the        Group I-T individuals improved noticeably in comparison to the        perfusion of the Group I-P individuals. Moreover, the perfusion        in Group I-P individuals showed a trend to deterioration.

The physiological study demonstrated an overall improvement in theperfusion and stress tolerance of Group I-T individuals when treatedwith pUVEK15. See Tables 1 and 2; FIGS. 1 and 2.

The histopathological study showed statistically significant differencesin numerical density, length density and mitotic index between bothsubgroups (inter-group non-paired comparisons). The Group I-Tindividuals presented higher mean values for these indexes when comparedto Group I-P individuals. See Tables 3, 4, 5 and 6; FIGS. 3, 4, 5, 6, 8,9, 10, 11, 12 and 13.

These results confirmed neovascular formation in vivo of myocardialtissue in the individuals treated with pUVEK15. Vascular formation andgrowth implies an increase in the number of cells taking part ofneovessels (endothelial and vascular smooth muscle cells). See FIGS. 12and 13. The administration of the inducing agent enhanced mitosis ofvascular cells in the individuals treated. The subgroup of individualstreated with pUVEK15 also showed a proportion of cardiomyocytes inmitotic process more than 5 times higher than the non-treated subgroup.See FIGS. 6, 8, 9, 10 and 11; Table 6.

Angiogenesis or other adverse side effects were not detected in theperipheral tissues of the individuals treated with pUVEK15.

2. Presence and Transcription of the VEGF Plasmid

Molecular studies showed presence of plasmid DNA in injected myocardialtissue of all Group II individuals (PCR technique). Plasmid DNA encodingfor VEGF was found in the injected myocardial tissue of the Group II-Tindividuals. Placebo plasmid DNA was found in the injected myocardialtissue of the Group II-P individuals.

A positive RT-PCR product for pUVEK15 was detected in the injectedmyocardial tissue of the Group II-T individuals at 3 (n=1/2), 10 (n=2/2)and 16 (n=1/2) days post-treatment. See FIG. 7. No RT-PCR product forpUVEK15 was detected in inject myocardial tissue at 35 days (n=2) afterpUVEK15 injection and in myocardial tissue receiving plasmid devoid ofgene (Group II-P).

A transcription curve (presence of mRNA) showing a peak by day 10post-injection of pUVEK15 was obtained in the Group II-T individuals.See FIG. 7. Presence of mRNA in group II-P was negative.

TABLE 1 Stress Tolerance Index Pre-treatment (1) Post-treatment (2) Pvalue Mean σ Mean σ (1) vs (2) Group I-P −0.6 2.2 −1.2 1.3 0.9 Group I-T−3.1 2.2 3.8 1.3 <0.01 P value 0.42 <0.02 I-T vs I-P

TABLE 2 Perfusion Improvement Index Mean σ Group I-P −0.6 2.6 Group I-T6.9 2.6 P value 0.058 I-T vs I-P

TABLE 3 Numerical Density Index (8-50 μm) Mean σ Group I-T 1 0.1 GroupI-P 0.6 0.1 P Value <0.02 I-T vs I-P

TABLE 4 Length Density Index (8-50 μm) Mean σ Group I-T 2.4 0.4 GroupI-P 1.3 0.3 P Value <0.02 I-T vs I-P

TABLE 5 Length Density Index (8-30 μm) Mean σ Group I-T 1 0.1 Group I-P0.6 0.1 P Value <0.02 I-T vs I-P

TABLE 6 Mitotic Index Mean σ Group I-T 187.1 49.6 Group I-P 35.4 9.1 PValue <0.04 I-T vs I-P

Example VI

Plasmids as above have also been introduced into sheep suffering fromacute myocardial infaction, and myocardiogenesis has been observed. Themethods in this study were adapted from the methods used in thepreceding Examples.

REFERENCES

The following references are referred to in abbreviated bibliographicform above.

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Multiple protein forms are encoded through alternative exon    splicing. J Biol Chem. 1991 Jun. 25; 266(18):11947-54.-   Robinson C J, Stringer S E. The splice variants of vascular    endothelial growth factor (VEGF) and their receptors. J Cell Sci.    2001 March; 114(Pt 5):853-65.-   Muller Y A, Christinger H W, Keyt B A, de Vos A M. The crystal    structure of vascular endothelial growth factor (VEGF) refined to    1.93 A resolution: multiple copy flexibility and receptor binding.    Structure. 1997 Oct. 15; 5(10):1325-38.-   McDonald N Q, Hendrickson W A. A structural superfamily of growth    factors containing a cystine knot motif Cell. 1993 May 7;    73(3):421-4.-   Murray-Rust J, McDonald N Q, Blundell T L, Hosang M, Oefner C,    Winkler F, Bradshaw R A. Topological similarities in TGF-beta 2,    PDGF-BB and NGF define a superfamily of polypeptide growth factors.    Structure. 1993 Oct. 15; 1(2):153-9.-   Sun P D, Davies D R. The cystine-knot growth-factor superfamily.    Annu Rev Biophys Biomol Struct. 1995; 24:269-91.-   Wiesmann C, Fuh G, Christinger H W, Eigenbrot C, Wells J A, de Vos    A M. Crystal structure at 1.7 A resolution of VEGF in complex with    domain 2 of the Flt-1 receptor. Cell. 1997 Nov. 28; 91(5):695-704.-   Fairbrother W J, Champe M A, Christinger H W, Keyt B A, Starovasnik    M A. Solution structure of the heparin-binding domain of vascular    endothelial growth factor. Structure. 1998 May 15; 6(5):637-48.-   Potgens A J, Lubsen N H, van Altena M C, Vermeulen R, Bakker A,    Schoenmakers J G, Ruiter D J, de Waal R M. Covalent dimerization of    vascular permeability factor/vascular endothelial growth factor is    essential for its biological activity. Evidence from Cys to Ser    mutations. J Biol Chem. 1994 Dec. 30; 269(52):32879-85.-   Keck P J, Hauser S D, Krivi G, Sanzo K, Warren T, Feder J, Connolly    D T. Vascular permeability factor, an endothelial cell mitogen    related to PDGF. Science. 1989 Dec. 8; 246(4935):1309-12.-   Siemeister G (a), Marme D, Martiny-Baron G. The alpha-helical domain    near the amino terminus is essential for dimerization of vascular    endothelial growth factor. J Biol Chem. 1998 May 1;    273(18):11115-20.-   Peretz D, Gitay-Goren H, Safran M, Kimmel N, Gospodarowicz D,    Neufeld G. Glycosylation of vascular endothelial growth factor is    not required for its mitogenic activity. Biochem Biophys Res Commun.    1992 Feb. 14; 182(3):1340-7.-   Claffey K P, Senger D R, Spiegelman B M. Structural requirements for    dimerization, glycosylation, secretion, and biological function of    VPF/VEGF. Biochim Biophys Acta. 1995 Jan. 5; 1246(1):1-9.-   Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth    factor. Endocr Rev. 1997 February; 18(1):4-25.-   Keyt B A, Nguyen H V, Berleau L T, Duarte C M, Park J, Chen H,    Ferrara N. Identification of vascular endothelial growth factor    determinants for binding KDR and FLT-1 receptors. Generation of    receptor-selective VEGF variants by site-directed mutagenesis. J    Biol Chem. 1996 Mar. 8; 271(10):5638-46.-   Siemeister G (b), Schirner M, Reusch P, Barleon B, Marme D,    Martiny-Baron G. An antagonistic vascular endothelial growth factor    (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation    and proliferation of human endothelial cells. Proc Natl Acad Sci    USA. 1998 Apr. 14; 95(8):4625-9.-   Houck K A, Leung D W, Rowland A M, Winer J, Ferrara N. Dual    regulation of vascular endothelial growth factor bioavailability by    genetic and proteolytic mechanisms. J Biol Chem. 1992 Dec. 25;    267(36):26031-7.-   Plouet J, Moro F, Bertagnolli S, Coldeboeuf N, Mazarguil H, Clamens    S, Bayard F. Extracellular cleavage of the vascular endothelial    growth factor 189-amino acid form by urokinase is required for its    mitogenic effect. 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Deposit

Plasmid pUVEK15 was deposited on Nov. 13, 2000, under access number DSM13833 at the DSMZ—Deutsche Sammlung von Mikroorganismen andZellkulturen, Mascheroder Weg 1B, D-38124 Braunschweig, Federal Republicof Germany.

The present invention has been described in some detail and exemplifiedto facilitate its understanding and reproducibility. Certain changes inthe form and detail can be made by anyone skilled in the art withoutdeparting from the true object and scope of the claims of the presentinvention. The disclosure of all applications, patents and publicationcited above and in the figures are hereby incorporated by reference intheir entirety.

We claim:
 1. A method for inducing mitosis and/or proliferation of aperipheral striated muscle cell in a tissue comprising peripheralstriated muscle cells, in a subject in need thereof, comprisingadministering directly to the cell or tissue a dose of a polynucleotidethat encodes the vascular endothelial growth factor, VEGF 1-165, whereinthe coding sequence is operably linked to a CMV promoter and is in aplasmid vector, the dose being in a sufficient amount to induce mitosisand/or proliferation of a peripheral striated muscle cell in a tissuecomprising striated muscle cells, wherein the dose is equivalent to theat least about 0.04 mg/kg that, when administered to a subject in needof the induction of cardiomyogenesis, induces cardiomyogenesis.
 2. Themethod of claim 1, wherein the VEGF 1-165 has the amino acid sequence:(SEQ ID NO: 1) Ala Pro Met Ala Glu Gly Gly Gly Gln AsnHis His Glu Val Val Lys Phe Met Asp ValTyr Gln Arg Ser Tyr Cys His Pro Ile GluThr Leu Val Asp Ile Phe Gln Glu Tyr ProAsp Glu Ile Glu Tyr Ile Phe Lys Pro SerCys Val Pro Leu Met Arg Cys Gly Gly CysCys Asn Asp Glu Gly Leu Glu Cys Val ProThr Glu Glu Ser Asn Ile Thr Met Gln IleMet Arg Ile Lys Pro His Gln Gly Gln HisIle Gly Glu Met Ser Phe Leu Gln His AsnLys Cys Glu Cys Arg Pro Lys Lys Asp ArgAla Arg Gln Glu Asn Pro Cys Gly Pro CysSer Glu Arg Arg Lys His Leu Phe Val GlnAsp Pro Gln Thr Cys Lys Cys Ser Cys LysAsn Thr Asp Ser Arg Cys Lys Ala Arg GlnLeu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg.


3. The method of claim 1, wherein the mitosis and/or proliferation islocalized.
 4. The method of claim 1, wherein the tissue comprisingstriated muscle cells is induced in normoperfused tissue.
 5. The methodof claim 1, wherein the tissue comprising striated muscle cells isinduced in ischemic tissue.
 6. The method of claim 1, wherein the cellor tissue is eukaryotic.
 7. The method of claim 1, wherein the cell ortissue is mammalian.
 8. The method of claim 1, wherein the cell ortissue is pig, rabbit, sheep or human.
 9. The method of claim 1, whereinthe cell or tissue is human.
 10. The method of claim 1, wherein thepolynucleotide is a genomic DNA, a cDNA, or a messenger RNA.
 11. Themethod of claim 10, wherein the polynucleotide encodes the polypeptiderepresented by SEQ ID NO:
 1. 12. The method of claim 11, wherein thepolynucleotide is a cDNA.
 13. The method of claim 1, wherein thepolynucleotide is administered to the cell or tissue in a liposome. 14.The method of claim 1, wherein the subject exhibits complications ofdiabetes.
 15. The method of claim 14, wherein the complications areperipheral artery disease (PAD), diabetic neuropathy, vasa-vasorumdisease and/or severe limb ischemia.
 16. The method of claim 1, whereinthe subject has PAD and/or limb ischemia.
 17. The method of claim 16,wherein the limb ischemia is severe limb ischemia.
 18. The method ofclaim 1, wherein the subject is human.
 19. The method of claim 1,wherein the polynucleotide is in the form of a pharmaceuticalcomposition.
 20. The method of claim 1, wherein the dose ofpolynucleotide is administered multiple times.